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Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice

Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung... www.nature.com/cddiscovery ARTICLE OPEN Co-targeting BCL-X and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice 1,2,3✉ 4,5 6 3 3 7 7 Sajid Khan , Patrick Kellish , Nick Connis , Dinesh Thummuri , Janet Wiegand , Peiyi Zhang , Xuan Zhang , 3,8,9 3 1,3 10 11 10 7 Vivekananda Budamagunta , Nan Hua , Yang Yang , Umasankar De , Lingtao Jin , Weizhou Zhang , Guangrong Zheng , 12 6 4 13 1,2,3 Robert Hromas , Christine Hann , Maria Zajac-Kaye , Frederic J. Kaye and Daohong Zhou © The Author(s) 2023 Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-X and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on- target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-X degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co- dependent on BCL-X and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-X /MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-X degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-X and MCL-1. Cell Death Discovery (2023) 9:1 ; https://doi.org/10.1038/s41420-022-01296-8 INTRODUCTION thus an urgent and unmet need to find newer treatment Small-cell lung cancer (SCLC) is a difficult-to-treat subtype of strategies to effectively treat SCLC. pulmonary carcinomas with a 5-year survival rate of ~5% and SCLC has historically been treated as a homogeneous disease. median survival of less than a year [1, 2]. First-line treatment for However, it has been recently demonstrated that inter- and intra- SCLC with combinations of a platinum-based agent (cisplatin or tumoral heterogeneity occurs in SCLC, is primarily responsible for carboplatin) and etoposide (a topoisomerase-II inhibitor) has reduced therapeutic efficacy and resistance [6–9]. Since unique remained largely unchanged for almost 35 years. Recently, a triplet resistance mechanisms occur in response to specific therapies, of atezolizumab (a PD-L1 antibody), carboplatin, and etoposide specific oncoproteins and/or pathways need to be defined and was approved as first-line therapy for extensive-stage SCLC but targeted for effectively treating SCLC. Aberrant expression of anti- provides only a moderate (~2 months) increase in overall survival apoptotic BCL-2 family proteins has also been shown to account compared to chemotherapy alone [3, 4]. More importantly, the for intratumoral heterogeneity and therapeutic resistance in SCLC SCLC tumors are initially responsive to chemotherapy, however, [6, 10]. This has been therapeutically exploited with a dual BCL-X / relapse occurs in a majority (>80%) of cases, and there is no BCL-2 inhibitor ABT263 (navitoclax) that shows high efficacy in effective second-line treatment for relapsed SCLC [4, 5]. There is preclinical models of SCLC [11, 12]. Unfortunately, the single-agent 1 2 Department of Biochemistry & Structural Biology, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Mays Cancer Center, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Department of Pharmacodynamics, College of Pharmacy, University of Florida, 4 5 Gainesville, FL, USA. Department of Anatomy & Cell Biology, College of Medicine, University of Florida, Gainesville, FL, USA. Department of Pediatrics, College of Medicine, 6 7 University of Florida, Gainesville, FL, USA. Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA. Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, USA. Genetics and Genomics Graduate Program, Genetics Institute, College of Medicine, University of Florida, 9 10 Gainesville, FL, USA. Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA. Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA. Department of Molecular Medicine, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Department of Medicine, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Division of Hematology and Oncology, Department of Medicine, College of Medicine, University of Florida, Gainesville, FL, USA. email: khans10@uthscsa.edu Received: 30 September 2022 Revised: 20 December 2022 Accepted: 22 December 2022 Official journal of CDDpress 1234567890();,: S. Khan et al. Fig. 1 The SCLC cell lines show differential survival dependence on BCL-2 family proteins. a The mRNA expression of BCL2 family anti- apoptotic genes as obtained from U Cologne study via cBioPortal. The mRNA expressions are depicted as Log2 (value + 1) from the RNA sequencing (RNA seq) dataset (n = 81 patient samples). b The EC values of A1155463 (a selective BCL-X inhibitor), venetoclax (a selective 50 L BCL-2 inhibitor), S63845 (a selective MCL-1 inhibitor), navitoclax (a BCL-X /BCL-2 dual inhibitor) and DT2216 (a selective BCL-X PROTAC L L degrader) were derived from % viability of indicated human SCLC cell lines after they were incubated with increasing concentrations of these inhibitors for 72 h. The data are presented from a single experiment performed in three replicate cell cultures. Similar results were obtained in two additional experiments performed with H146 and H378, and one additional independent experiment performed with H187, H211, and H209 cell lines. When an inhibitor showed an EC of ≤1 μM in any given cell line, the cell line was considered as sensitive to that inhibitor and dependent on its target BCL-2 family protein. efficacy of navitoclax against SCLC in phase-II clinical trials was limiting toxicities. We have devised a safer strategy to target these limited [13] because of the intratumoral heterogeneity and tumors as we found that BCL-X /MCL-1 co-dependent SCLC cells dependence of some of the cells within a tumor on other BCL-2 can be synergistically killed with a combination of DT2216 (a family proteins such as MCL-1. More importantly, the clinical use selective BCL-X PROTAC degrader) and AZD8055 (an mTOR of ABT263 is hampered by dose-limiting severe thrombocytopenia inhibitor). This was achieved by selective degradation of BCL-X caused by BCL-X inhibition [14, 15]. We are now able to safely and inhibition of MCL-1 expression in tumor cells with DT2216 and target BCL-X using proteolysis targeting chimeras (PROTACs, such AZD8055, respectively, therefore reducing on-target toxicity to as DT2216) without causing appreciable platelet toxicity (throm- platelets and other normal cells. In vivo, the combination of bocytopenia) as recently reported by our group [16–20]. DT2216 and AZD8055 strongly inhibited the growth of cell line- In this study, we aimed to develop a synergistic and safe derived and patient-derived xenograft (PDX) tumors and reduced therapeutic strategy for SCLC. This was achieved first by profiling tumor burden as well as increased survival of a murine Rb1/p53/ the heterogeneity of survival dependence of a panel of SCLC cell p130 SCLC genetically engineered mouse (GEM) model without lines on the BCL-2 family anti-apoptotic proteins by BH3 mimetic causing appreciable thrombocytopenia or other normal tissue screening, where we used BH3 mimetics to target specific BCL-2 injuries. anti-apoptotic proteins followed by measurement of cell viability [21]. We defined a subset of SCLC cell lines that are co-dependent on BCL-X and MCL-1 for survival and focused on testing the RESULTS ability to target these cell lines effectively and safely. Currently, BH3 mimetic screening identifies DT2216 sensitive and such tumors cannot be safely targeted with available inhibitors. resistant SCLC cells in vitro For example, MCL-1 inhibition causes severe cardiotoxicity, and We first explored the mRNA expression of anti-apoptotic BCL-2 co-targeting BCL-X and MCL-1 with commercially available family genes in patient tumors via cBioPortal [25]. These data inhibitors further exacerbates normal tissue injury and causes showed that BCL2, BCL2L1 (BCL-X coding gene), MCL1, and lethality [22–24]. Therefore, we need to develop an alternate BCL2A1 (BFL-1 coding gene) are abundantly expressed in most strategy to selectively suppress BCL-X and MCL-1 in co- SCLC patients’ tumors (Fig. 1a). Unfortunately, no healthy lung dependent SCLC tumors to avoid their on-target and dose- tissue transcriptomics data were available for a side-by-side Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 2 A subset of SCLC cell lines is co-dependent on BCL-X and MCL-1. a Viability graphs of representative SCLC cell lines showing dependence on individual or a combination of BCL-2 family anti-apoptotic proteins. The data are presented as mean ± SD from a single experiment (n = 3 replicate cell cultures). Similar results were obtained in one additional independent experiment performed with H209 and H211, and two additional independent experiments performed with H378 cells. b, c Viability of H378 and H1048 SCLC (b) and NHBE (c) cells after they were treated with increasing concentrations of DT2216 (DT) or S63845 (S) or their combination (1:1 ratio) for 72 h. EC and CI values are shown. The data are presented as mean ± SD from a representative experiment (n = 3 replicate cell cultures). Similar results were obtained in one additional independent experiment. comparison to show whether or not these genes were over- these may co-depend on a combination of either BCL-X /MCL-1 or expressed in SCLC. Therefore, to establish the importance of these BCL-2/MCL-1 or BCL-X /BCL-2/MCL-1 (Fig. 1b). These results BCL-2 family of anti-apoptotic proteins in SCLC, we systematically suggest that BCL-X is a key pro-survival protein in SCLC and evaluated the effects of inhibitors of BCL-2 family anti-apoptotic DT2216 might have a broad-spectrum antitumor activity than a proteins, i.e., A1155463 (a selective BCL-X inhibitor), venetoclax (a BCL-X inhibitor in killing BCL-X -dependent SCLC cells. L L L selective BCL-2 inhibitor), S63845 (a selective MCL-1 inhibitor) and navitoclax (a BCL-X /BCL-2 inhibitor) on the viability of a panel of A combination of MCL-1 inhibitor with BCL-X inhibitor or L L 20 SCLC cell lines, which was named as the BH3 mimetic screening PROTAC is not selective to tumor cells [21]. The cell lines with EC < 1 µM for a particular inhibitor were In the BH3 mimetic screening, SCLC cell lines showed varying considered sensitive. SCLC cell lines showed differential depen- responses to the inhibitors of different BCL-2 family anti-apoptotic dence on different BCL-2 family anti-apoptotic proteins, with a proteins. Based on these dependencies, we divided SCLC cell lines majority of them depending on BCL-X (9/20, 45%), while some into different categories; 1) primarily dependent on BCL-X (such L L depended on a combination of BCL-X and BCL-2 (3/20, 15%), and as H1963), 2) mainly dependent on BCL-2 (such as H889), 3) mostly only a few depended on BCL-2 (2/20, 10%) or MCL-1 (2/20, 10%), dependent on MCL-1 (such as H209), 4) BCL-X and BCL-2 co- as indicated by their sensitivity to these specific inhibitors (Fig. 1b). dependent (such as H1059), 5) BCL-X and MCL-1 co-dependent One cell line (H211) was found to be sensitive to all of these (such as H378), and 6) BCL-X , BCL-2, and MCL-1 dependent (such inhibitors indicating that all three, i.e., BCL-X , BCL-2, and MCL-1 as H211) (Fig. 2a). When two or more proteins were needed to be are important for its survival. These findings are corroborated by simultaneously inhibited in order to sensitize a particular cell line, the transcriptomic profiling suggesting that BCL-X is one of the we called it co-dependent on those proteins. For example, H1059 highly expressed BCL-2 family anti-apoptotic proteins, and there- cells were only sensitive upon simultaneous inhibition of BCL-X fore, a promising therapeutic target in SCLC tumors [10–12]. and BCL-2 using navitoclax, therefore, we categorized them as Further, we evaluated the activity of our BCL-X PROTAC degrader BCL-X and BCL-2 co-dependent. Similarly, H378 cells were L L DT2216 on the viability of these SCLC cell lines where 50% (10/20) sensitive to simultaneous inhibition of BCL-X and MCL-1, and cell lines were found to be sensitive to DT2216 with an EC of therefore, categorized as BCL-X /MCL-1 co-dependent. These 50 L <1 µM. All of the cell lines that were found to be sensitive to the results indicate that SCLC cells are heterogeneous in their BCL-X inhibitor were sensitive to DT2216 as well. Interestingly, the dependencies on BCL-2 anti-apoptotic proteins. H1059 cell line was not sensitive to the BCL-X inhibitor but was Next, we focused on targeting BCL-X and MCL-1 co-dependent L L found to be sensitive to DT2216. Six of the cell lines did not cells, as these types of SCLC tumors are highly resistant to respond to any of these inhibitors or DT2216 suggesting that chemotherapy and cannot be safely eradicated with the currently Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 3 mTOR inhibitor AZD8055 selectively suppresses MCL-1 expression in tumor cells. a, b Immunoblot analyses of MCL-1, BCL-X and L, BCL-2 in SCLC H378 and H1048 cell lines (a) and NHBE cells (b) after they were treated with indicated agents (1 µM each) for 24 h. Normalized densitometric values for MCL-1 blots are shown underneath. c, d Immunoblot analysis of mTOR substrates (p-4EBP1 and p-S6), MCL-1, BCL-X , and BCL-2 after 24 h treatment with indicated concentrations of AZD8055 in H378 and H1048 SCLC cell lines (c) and NHBE cells (d). The uncropped immunoblot images related to this figure are provided in the “Supplemental Material” file. available inhibitors of BCL-2 family proteins because the inhibitor that primarily targets CDK9), AZD8055 (an ATP- combination of BCL-X and MCL-1 inhibitors causes severe normal competitive catalytic mTORC1/2 inhibitor), TD-19 (a cancerous tissue toxicities and lethality in mice [23, 24, 26]. This was inhibitor of protein phosphatase 2A inhibitor) and piperazine (a confirmed by our study showing that a combination of DT2216 or protein phosphatase 2A [PP2A] activator). All of these therapeutic A1155463 with MCL-1 inhibitor (S63845) synergistically kills not agents have previously been shown to suppress MCL-1 expression only tumor cells (Fig. 2b), but also normal cells from different through different mechanisms [10, 26–29]. Here, we aimed to tissue origins such as human bronchial epithelial cells (NHBE), determine whether any of these agents could selectively suppress colon fibroblasts (CCD-18Co) and lung fibroblasts (WI38) (Fig. 2c; MCL-1 expression in tumor cells while having minimal/no supplementary Fig. 1a). Of note, S63845 treatment alone was also significant effect on the expression of MCL-1 in normal cells. In highly toxic to NHBE cells, which indicates that these cells agreement with previous reports, doxorubicin, SNS-032 and primarily depend on MCL-1 for survival. These findings do not AZD8055 caused significant suppression of MCL-1 expression in support using the combination of a BCL-X inhibitor/PROTAC and all tested SCLC cell lines (Fig. 3a; Supplementary Fig. 1b). Among an MCL-1 inhibitor to treat BCL-X and MCL-1 co-dependent SCLC these inhibitors tested, only AZD8055 showed minimal effect on in the clinic. the expression of MCL-1 in normal cells (Fig. 3b; Supplementary Fig. 1c). None of these agents, except SNS-032, significantly altered DT2216+AZD8055 combination synergistically kills BCL-X / BCL-X or BCL-2 levels in both tumor cells as well as normal cells. L L MCL-1 co-dependent SCLC cells through degradation of BCL- Further, we analyzed the dose-dependent effect of AZD8055 on X and suppression of MCL-1 expression, respectively, in a the expression of MCL-1, BCL-X and BCL-2 in H378 and H1048 L L, tumor cell-selective manner SCLC and NHBE normal cells. AZD8055 caused dose-dependent Next, we sought to identify a strategy to selectively suppress suppression of MCL-1 expression in tumor cells without any MCL-1 expression in SCLC cells by rationally targeting certain observable effect in normal cells. As expected, AZD8055 had no tumorigenic proteins/pathways. We screened several clinical-stage observable effects on the expression of BCL-X and BCL-2 in all the compounds and FDA-approved drugs including doxorubicin (a tested cell lines. Of note, BCL-2 expression was not detected in topoisomerase II inhibitor and a commonly used chemotherapeu- NHBE cells via immunoblotting. Also, AZD8055 dose-dependently tic), SNS-032 (a promiscuous cyclin-dependent kinase [CDK] inhibited activation of the mTOR downstream targets, i.e., p-4EBP1 Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 4 DT2216+AZD8055 combination synergistically kills BCL-X /MCL-1 co-dependent SCLC cells through disruption of MCL-1/BIM interaction. a, b Viability of H378 and H1048 SCLC (a) and NHBE (b) cells after they were treated with increasing concentrations of DT2216 (DT) or AZD8055 (AZD) or their combination (1:1 ratio) for 6 days. EC and CI values are shown. The data presented in a and b are mean ± SD from a representative experiment (n = 3 replicate cell cultures). Similar results were obtained in two additional independent experiments. c, d Immunoblot analyses of full-length PARP (fPARP) and cleaved PARP (cPARP) in SCLC H378, H1048 (c), and NHBE (d) cell lines after they were treated with 1 µM each of DT2216 or AZD8055 or their combination for 24 h. e Immunoprecipitation analysis of MCL-1 followed by immunoblotting of BIM and MCL-1 in H1048 cells after they were treated with 1 µM each of DT2216 and/or AZD8055 for 24 h. Immunoblot analyses of BIM, MCL-1, and BCL-X in input samples are shown on the right. The uncropped immunoblot images related to this figure are provided in the “Supplemental Material” file. and p-S6, in both SCLC and NHBE cells indicating a desired on- inhibitor. Everolimus is FDA-approved for use as an immunosup- target effect (Fig. 3c, d). pressant and for treating certain tumors. We wondered whether Since AZD8055 was found to selectively suppress MCL-1 everolimus can also synergistically kill BCL-X /MCL-1 co- expression in SCLC cells, we next evaluated it in combination dependent SCLC cells when combined with DT2216. Indeed, the with DT2216 on the viability of BCL-X /MCL-1 co-dependent SCLC combination of everolimus and DT2216 synergistically reduced cells as well as normal cells. Out of 11 SCLC cell lines that were the viability of H378 and H1048 cells, as well as more effectively resistant to BCL-X inhibitor (A1155463) alone in Fig. 1b, induced apoptosis compared to individual agents in H1048 cells we selected the five most resistant cell lines to test against (Supplementary Fig. 4a, b). However, unlike AZD8055, everolimus the combination of BCL-X inhibitor and MCL-1 inhibitor. Out of alone was less effective in inhibiting the viability of H1048 cells, these five, three cell lines (H378, H1048, and DMS53) were found which might be because it caused even lesser apoptosis to be sensitive to dual BCL-X and MCL-1 inhibition. Further, we compared to AZD8055 (Supplementary Fig. 3b, 4b). This was used the two most sensitive (H378 and H1048) cell lines to test the accompanied by a significant reduction in MCL-1 expression in combination of DT2216 and AZD8055. The DT2216 + AZD8055 H1048 cells after everolimus treatment (Supplementary Fig. 4c). combination was found to synergistically inhibit the viability of These results suggest that the tumor-specific inhibition of only H378 and H1048 SCLC cells, but not normal NHBE and CCD-18Co mTORC1 is sufficient for the suppression of MCL-1 expression. cells, as assessed by CI values at EC (concentration with 75% Also, mTOR seems to differentially regulate MCL-1 in tumor and viability loss) and EC (concentration with 90% viability loss) normal cells, therefore we could not see MCL-1 downregulation using Chou-Talalay method [30] (Fig. 4a, b; Supplementary Fig. even after efficient mTOR inhibition in NHBE cells. 1d). Notably, AZD8055 treatment alone led to partial inhibition of To gain further insights into the mechanism of synergistic activity H1048, NHBE, and CCD-18Co cell viability which mainly attributed of DT2216+ AZD8055, we performed a co-immunoprecipitation to its antiproliferative effects. Importantly, the combination of (co-IP) analysis of MCL-1 and BIM because BIM acts as a BH3-only DT2216 and AZD8055 caused a complete loss of viability of tumor pro-apoptotic protein that can trigger apoptosis by activating the cells, but not normal cells. Therefore, the combination effects were apoptotic effectors BAX and BAK and the sequestration of BIM by synergetic in tumor cells, but not in normal cells, as indicated by MCL-1 or BCL-X inhibits apoptosis [31]. Since BIM is known to bind the CI values. DT2216 treatment induced a dose-dependent BCL- both BCL-X and MCL-1, we hypothesized that the degradation of X degradation in H1048 cells as expected (Supplementary Fig. 2). BCL-X with DT2216 may lead to an increased association of BIM L L Since 1 µM of DT2216 was found to completely deplete BCL-X ,we with MCL-1, and co-treatment with DT2216+ AZD8055 may disrupt selected this concentration for further in vitro experiments. In this interaction. In line with our hypothesis, we found that DT2216 addition, we found that the combination of DT2216 and AZD8055 treatment increases the BIM/MCL-1 association which was disrupted was more effective compared to individual agents in inhibiting when the cells were treated with the DT2216+ AZD8055 combina- H1048 cell survival and inducing apoptosis by performing the tion (Fig. 4e). Interestingly, increased BIM binding seems to stabilize long-term clonogenic assay and flow cytometric analysis, respec- MCL-1 upon DT2216 treatment, and the MCL-1 levels are tively (Supplementary Fig. 3a, b). Furthermore, the suppressed in the combination-treated cells. Since BCL-X inhibition DT2216 + AZD8055 combination profoundly induced PARP clea- has been shown to increase MCL-1 levels in some cancer cell lines as vage compared to individual agents in tumor cells, but not in acompensatorymechanism [32]. So, the increased MCL-1 levels NHBE cells, which again confirmed the tumor cell-selectivity of the upon DT2216 treatment may be due to either BIM-induced combination (Fig. 4c, d). stabilization and/or a compensatory mechanism that needs further Unlike AZD8055 which inhibits both mTORC1 and mTORC2, exploration. everolimus is a rapamycin derivative and a selective mTORC1 Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 5 The combination of DT2216 and AZD8055 has stronger antitumor responses in H1048 xenograft and SCLC PDX models. a, b Tumor volume (a) and mouse body weight (b) change in H1048 xenografts after treatment with vehicle (Veh), DT2216 (DT, 15 mg/kg/q4d, i.p.), AZD8055 (AZD, 16 mg/kg/5x week, p.o.) or a combination of DT and AZD. Data are presented as mean ± SEM (n = 5 mice in Veh and single- treatment groups, and six mice in combination group at the start of treatment). c Platelet counts 24 h after the first dose of DT2216 (DT, 15 mg/kg/q4d, i.p.) and/or AZD8055 (AZD, 16 mg/kg/5x week, p.o.) in H1048 xenograft mice from a separate experiment (n = 7 mice in Veh and single-treatment groups, and eight mice in combination group). d Immunoblot analysis of indicated proteins in H1048 xenograft tumors at the end of treatments as in a (n = 3 mice per group). e Densitometry of immunoblots shown in panel d (mean ± SEM). f, g Tumor volume (f) and mouse body weight (g) changes in LX47 PDX tumors after treatment with veh, DT2216 (DT, 15 mg/kg/q4d, i.p.) or AZD8055 (AZD, 16 mg/ kg/5x week, p.o.) or a combination of DT and AZD. Data are presented as mean ± SEM (n = 8 mice in single-treatment groups, and nine mice in Veh and combination groups at the start of treatment). Statistical significance in a, c, e, and f was determined by a two-sided unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. The uncropped immunoblot images related to this figure are provided in the “Supplemental Material” file. DT2216+AZD8055 combination synergistically inhibits tumor be clinically safe [16]. Therefore, a 25–30% reduction in total platelet growth in SCLC xenograft and PDX models count may likely not be of any clinical concern. Next, we confirmed To further assess the combined mTOR inhibition and BCL-X that DT2216 and AZD8055 led to a significant reductioninBCL-X L L degradation as a potential therapeutic strategy for BCL-X and and MCL-1 levels, respectively, which was associated with significant MCL-1 co-dependent SCLC, we first investigated the efficacy of inhibition of mTOR substrates (p-4EBP1 and p-S6) upon AZD8055 DT2216+ AZD8055 combination using the H1048 xenograft tumor treatment in H1048 tumor lysates. Interestingly, the combination model. In agreement with the in vitro results, DT2216 alone had no also caused a significant decrease in BCL-2 protein levels in these effect on H1048 tumor growth. Although AZD8055 alone showed tumor samples (Fig. 5d, e). some efficacy, the effect was not significant. On the other hand, the Since the PDX models better recapitulate human cancers in vivo, combination of DT2216 and AZD8055 led to significant inhibition of we next evaluated the effect of the combination of tumor growth (Fig. 5a). More importantly, the combination DT2216+ AZD8055 in the LX47-SCLC PDX model. We found that treatment appeared to be safe as indicated by no significant the combination significantly inhibited tumor growth in this PDX change in mouse body weights after the treatment (Fig. 5b). In model of SCLC, whereas single agents failed to have significant addition, we did not observe any gross organ toxicities upon effects(Fig. 5f). Again, we did not observe any significant body necropsy in mice treated with DT2216+ AZD8055. Furthermore, the weight loss with the combination treatment in these mice (Fig. 5g). combination caused a similar reduction (25–30%) in platelets as DT2216 alone, where platelet counts remained well above 2 × 10 DT2216+AZD8055 combination inhibits lung tumor growth per µL of blood (Fig. 5c). Notably, platelet counts above 2 × 10 per and increases mice survival in the Rb1/p53/p130 GEM model µL of blood in mice (equivalent to 5 × 10 per µL in humans) are not Next, we used the conditional-mutant Rb1/p53/p130 mouse model associated with a significant risk of hemorrhage and considered to of SCLC to test the efficacy of the DT2216+ AZD8055 combination. Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 6 The combination of DT2216 and AZD8055 has stronger antitumor responses as compared to individual agents in a GEM model of SCLC. a Experimental design of Rb1/p53/p130 GEM model. Mice were administered with 2.5 × 10 pfu/mouse of Ad5CMVCre-eGFP through nasal inhalation. Treatment was started 110 days post-infection and continued until day 150. Specifically, the mice were treated with vehicle (veh), AZD8055 (AZD, 16 mg/kg/5x week, p.o.) and AZD (16 mg/kg/5x week, p.o.) + DT2216 (DT, 15 mg/kg/q4d, i.p.) (n= 8, 8 and 7 mice in veh, AZD and combo groups, respectively). The figure was adapted from BioRender. b Kaplan–Meier survival analysis of mice as treated in a. The median survival time is shown on the right. At the end of the experiment (day 166), 2, 2, and 4 mice were alive in veh, AZD and DT + AZD groups, respectively. c Bouin’s-stained represented images of excised lungs from veh, AZD, and DT + AZD groups. The tumor nodules in the lungs are circled. As shown, the tumor nodules were considerably smaller in the lungs of combination-treated mice as compared to veh or AZD-treated mice. d The Number of tumor nodules was counted on both dorsal and ventral sides of the lungs and the average number of tumor nodules per mouse lungs in each group is shown (mean ± SEM, n = 7). Statistical significance was determined by a two-sided unpaired Student’s t test. **p < 0.01. e Hematoxylin and eosin (H&E) staining of representative lung sections in veh, DT, AZD, and DT + AZD groups from a separate experiment in which the mice were treated with veh, DT (15 mg/kg/q4d, i.p.), AZD (16 mg/kg/5x week, p.o.), and a combination of DT and AZD. The mice were euthanized 24 h after the last treatment. In these mice, Rb1, Tp53, and p130 tumor suppressor genes have mTOR inhibition in part via depleting tumor-infiltrating regulatory flanking lox sequences that are under the control of Cre T cells (TI-Tregs) as shown in our recent study [36]. Depletion of recombinase. These genes are deleted upon administration of TI-Tregs by DT2216 may stimulate antitumor immunity and Adenovirus (Ad5CMVCre-eGFP), specifically in the lung epithelial attenuate mTOR inhibition-induced immunosuppression and cells, and lead to tumor formation in the lungs [33, 34]. Since the spleen size reduction. Furthermore, we did not see any clinically Rb1/p53/p130 mouse tumors closely resemble human SCLC, it is a significant reduction in platelets and other blood cell counts in the suitable preclinical mouse model to evaluate newer therapeutics treatment cohorts (Supplementary Fig. 5b, c). Overall, these results against SCLC. We determined a mean tumor onset time of suggest that the DT2216+ AZ8055 combination may be a 110 days after administration of adenovirus titer of 2.5 × 10 pfu/ promising therapeutic strategy, particularly against BCL-X /MCL-1 mouse to induce the formation of small visible tumor nodules in co-dependent SCLC, and therefore this combination could the mouse lungs. Therefore, we started treating the mice after potentially be tested in clinical trials in the future. 110 days with DT2216 and/or AZD8055 for up to 150 days (Fig. 6a). We did not include the DT2216 alone group because DT2216 was not found to have any significant effect on tumor growth when DISCUSSION used alone in this model in a previous study as shown in Fig. 6e. SCLC shows high inter- and intra-tumoral heterogeneity which is Mice were euthanized, and the survival was recorded at the primarily responsible for the therapeutic resistance [6–9]. This humane endpoint when the body condition score of animals heterogeneity is in part attributed to an aberrant expression of declined [35]. At the end of the experiment i.e, 150 days after anti-apoptotic BCL-2 family proteins which are crucial for the treatment, 2/8 (25%), 2/8 (25%) and 4/7 (~57%) mice were alive in survival of SCLC cells [10–12]. In the present study, we first the vehicle, AZD8055, and combination-treatment groups, respec- established the survival dependence of SCLC cell lines on different tively. The median survival time was 131, 149.5, and >166 days in BCL-2 family anti-apoptotic proteins using BH3 mimetic screening. the vehicle, AZD8055, and the combination-treatment group, We found that most SCLC cell lines are dependent on BCL-X for respectively (Fig. 6b). We excised the lungs from these mice and their survival and are highly sensitive to a BCL-X inhibitor or our observed that the combination-treated lungs had much smaller recently developed BCL-X PROTAC DT2216 (now a clinical tumor nodules as compared to vehicle or AZD8055 treatments candidate in the phase-I trial) [16, 17]. On the other hand, some (Fig. 6c). Moreover, the number of total tumor nodules per lungs SCLC cell lines were found to be dependent on multiple co- was also significantly reduced in the combination-treated mice expressed BCL-2 family proteins such as concurrent BCL-X and (Fig. 6d). The histopathological staining in the lungs from a BCL-2, or concurrent BCL-X and MCL-1. BCL-X /BCL-2 co- L L separate experiment further evidenced the reduction in tumor dependent cell lines could be targeted with navitoclax, however, burden in the combination-treated mice (Fig. 6e). We did not see the clinical translation of navitoclax is hampered by dose-limiting any significant changes in the lungs, spleen, liver, or kidney on-target toxicity of thrombocytopenia [14, 15]. More importantly, weights after combination treatment (Supplementary Fig. 5a). The BCL-X /MCL-1 co-dependent tumor cells cannot be safely targeted reductions in spleen weight in AZD-treated mice might be with the currently available inhibitors because the combination of attributable to the immunosuppressive effect of mTOR inhibitors. BCL-X and MCL-1 inhibitors causes severe tissue damage and DT2216 might be able to reduce the immunosuppressive effect of lethality in mice [22–24]. Therefore, finding a strategy to Cell Death Discovery (2023) 9:1 S. Khan et al. selectively target BCL-X and MCL-1 in tumors without causing YAP1 [5, 7]. Each of these molecular subtypes shows distinct significant on-target toxicity was the primary goal of our current therapeutic vulnerabilities [8, 9]. For example, ASCL1-high subtype study for treating an important difficult-to-treat subset of SCLC. (classic or neuroendocrine (NE) SCLC) [7] has been shown to be Since systemic MCL-1 inhibition with currently available sensitive to BCL-2 inhibition with ABT263 [43]. Interestingly, we inhibitors leads to normal tissue toxicity and lethality when observed that most variant-SCLC cells (non-NE consisting of combined with a BCL-X inhibitor/PROTAC, we sought to identify a POU2F3 and/or YAP1 subtype) [7] were dependent on the co- strategy to selectively suppress MCL-1 expression in SCLC cells. expression of BCL-X and MCL-1. Although these subtypes are not After screening several compounds that target oncogenic path- being used to direct treatment decisions for SCLC at present, we ways known to upregulate MCL-1 [10, 26–29], we identified that expect that POU2F3 and/or YAP1 SCLC subtypes could be better AZD8055, a mTORC1/2 ATP-competitive inhibitor, and everolimus, targeted with a combination of DT2216 and an mTOR inhibitor. In a selective mTOR1 inhibitor, can selectively suppress MCL-1 this case, immunohistochemical or proteomics profiling of four of expression in SCLC cells but not in normal cells. In contrast, other these transcriptional regulators could be a better alternative to compounds such as doxorubicin and SNS-032 non-specifically BH3 profiling for stratifying patients who can specifically benefit downregulated MCL-1 in tumor cells as well as in normal cells. This from the DT2216 + mTOR inhibitor combination. In the current was correlated with high basal mTOR activation in MCL-1- study, we used AZD8055 as a proof of concept, but it would be dependent SCLC cell lines (data not shown). The mTOR activation suitable to use an FDA-approved mTOR inhibitor (such as has been shown to enhance cap-dependent MCL-1 translation, everolimus) in combination with DT2216 in clinical trials. and therefore, inhibition of mTOR suppresses MCL-1 at post- In conclusion, the findings from our in vitro and in vivo studies translational level [26, 28, 29, 37]. Of note, the combination of suggest that the combination of DT2216 + AZD8055 is synergistic DT2216 with AZD8055 or everolimus synergistically inhibited the and somewhat tumor-selective against the BCL-X /MCL-1 co- viability and induced apoptosis in BCL-X and MCL-1 co- dependent subset of SCLC and is tolerable in mice without dependent SCLC cell lines, but not the normal cells from different appreciable on-target toxicity and normal tissue injury. These tissue origins including lungs and colon. findings may have a high potential for near-term clinical Our further investigation using different in vivo SCLC models translation as the combination of DT2216 and an FDA-approved including conventional xenografts, PDX, and Rb1/p53/p130 GEM mTOR inhibitor (such as everolimus) can be rapidly evaluated in model also suggests that the combination of DT2216 and SCLC patients, given that DT2216 is already in the phase-I clinical AZD8055 can effectively inhibit the SCLC growth in mice. More trial (Identifier: NCT04886622). This kind of approach can help to importantly, the combination appeared to be safe as evidenced by effectively treat an important and difficult-to-treat subset of SCLC no significant decrease in mouse body weights as well as no patients in the near future. clinically significant reduction in different blood cells including platelets, and the absence of any observable tissue pathology. Interestingly, results using the SCLC GEM model, which closely MATERIALS AND METHODS recapitulates genetic heterogeneity found in human SCLC, Cell lines and culture All the SCLC cell lines, except DMS53 and DMS114, were obtained from the indicate that the combination of DT2216 + AZD8055 strongly original NCI-Navy Medical Oncology source supply [44]. DMS53 and inhibits tumor growth in the lungs and increased the survival of DMS114 SCLC cell lines, CCD-18Co normal colon fibroblasts, and WI-38 mice. The effectiveness of the combination in the GEM model was lung fibroblasts were purchased from the American Type Culture encouraging because the tumors in this model have been shown Collection (ATCC, Manassas, VA). SCLC cell lines were cultured in RPMI- to be highly resistant to cisplatin plus etoposide doublet 1640 medium (Cat. No. 22400–089, Thermo Fisher, Waltham, MA). CCD- chemotherapy [26]. 18Co and WI-38 cells were cultured in Dulbecco’s modified Eagle’s medium These findings have important clinical implications because (DMEM) (Cat. No. 12430-062, Thermo Fisher). The culture media were tumor-specific targeting of BCL-X and MCL-1 has high therapeutic supplemented with 10% heat-inactivated fetal bovine serum (FBS, Cat. No. value. Two prior studies have found that a combination of S11150H, Atlanta Biologicals, GA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pen-Strep, Cat. No. 15140122, Thermo Fisher). NHBE cells navitoclax and AZD8055 synergistically kills different tumor cells were purchased from Lonza (Cat. No. CC-2541, Basel, Switzerland), and from SCLC and BRAF/KRAS-mutated colorectal cancer [26, 28]. were cultured in Lonza’s bronchial epithelial cell growth medium (Cat. No. Moreover, a study from Dr. Christine Hann’s group has demon- CC-3170) with supplements and growth factors (CC-4175). The stocks of strated synergy between rapamycin and ABT737 (a predecessor NCI SCLC cell lines that we used were STR profiled by NIH, so the compound of navitoclax) against different PDX models of SCLC authenticity of the cell lines remained preserved. All cultures were [38]. In another study, the combined inhibition of BCL-X and confirmed for Mycoplasma negativity using the MycoAlert Mycoplasma mTOR was found to synergistically induce apoptosis in PIK3CA- Detection Kit (Cat. No. LT07–318). All the cell lines were maintained in a mutated breast cancer [37]. However, these studies did not aim to humidified incubator at 37 °C and 5% CO . comprehensively evaluate tumor-selectivity and on-target toxi- cities of this combination (especially in mouse models). Given that Chemical compounds BCL-X inhibitors including navitoclax cause thrombocytopenia, a DT2216 was synthesized in Dr. Guangrong Zheng’s laboratory (University of combination of navitoclax with AZD8055 (or another mTOR Florida, Gainesville, FL) according to the previously described protocol [16]. inhibitor) may not be clinically feasible [14, 15]. In contrast, by AZD8055 (Cat. No. HY-10422) and everolimus (Cat. No. HY-10218) were combining AZD8055 with platelet-sparing BCL-X degrader purchased from MedChemExpress (Monmouth Junction, NJ). A1155463 (Cat. No. S7800), ABT199 (Cat. No. S8048), S63845 (Cat. No. S8383), and DT2216, we achieved tumor-selective targeting of both MCL-1 ABT263 (Cat. No. S1001) were purchased from SelleckChem (Houston, TX). and BCL-X , respectively, without significant on-target toxicity both in cell culture and in mice. This approach can also be applied to other tumor types which are co-dependent on MCL-1 and BCL- Cell viability assays X [39, 40]. One limitation of our in vitro BH3 mimetic screening is The cell viability was measured by MTS assay according to the manufacturer’s protocol (Cat. No. G-111, Promega, Madison, WI) and as that it requires live cells, so it cannot be used to stratify patients described previously [16, 39]. EC values were determined using who will benefit from the combination. In that case, a dynamic GraphPad Prism software (GraphPad Software, La Jolla, CA). BH3 profiling assay would be more useful as demonstrated previously [41, 42]. More recently, SCLC tumors have been classified into four BH3 mimetic screening The screening was performed to determine the survival dependence of molecular subtypes based on the relative expression of four SCLC cells on different BCL-2 family anti-apoptotic proteins [21]. The cells transcriptional regulators, i.e., ASCL1, NEUROD1, POU2F3, and Cell Death Discovery (2023) 9:1 S. Khan et al. were treated in 96-well plates with either a specific inhibitor of BCL-2 were treated with vehicle, AZD8055 (16 mg/kg, 5 days a week, p.o.), and a family anti-apoptotic proteins such as A1155463 (selective BCL-X combination of DT2216 (15 mg/kg, q4d, i.p.) and AZD8055 (n = 7–8 mice inhibitor), venetoclax (selective BCL-2 inhibitor), and S63845 (selective per group). Survival events were scored when the body condition score (as MCL-1 inhibitor), or a non-specific inhibitor such as navitoclax (BCL-X /BCL- defined by the American Association for Laboratory Animal Science) of 2 dual inhibitor). Thereafter, the viability was assessed using the MTS assay animals declined or per absolute survival events [35]. The lungs were as described above. excised upon euthanizing the individual mice, stained in Bouins’ solution, and photographed as described previously [45, 46]. The number of visible tumor nodules was counted on the surface of each pair of lungs and is Co-immunoprecipitation presented as the average number of tumor nodules/lungs. Cell pellets were lysed in the Pierce IP lysis buffer (Cat. No. 87787; Thermo Fisher) supplemented with protease and phosphatase inhibitors as described previously [16, 39]. The supernatants were collected and Statistical analysis precleared by incubating with 1 µg of mouse anti-IgG (Cat. No. sc-2025; All the graphs presented in this manuscript were made and statistical Santa Cruz Biotechnology [SCB], Dallas, TX) and 20 µL of protein A/G-PLUS analyses were performed using the GraphPad Prism-9 software. A two- agarose beads (Cat. No. sc-2003; SCB) for 30 min at 4 °C. The supernatants sided unpaired Student’s t test was used for comparisons between the containing 1 mg of protein were incubated with 2 µg of anti-MCL-1 (Cat. means of the two groups. P < 0.05 was considered to be statistically No. sc-12756; SCB) or anti-IgG antibody overnight followed by incubation significant. The combination index (CI) was calculated using Compusyn with 25 µL protein A/G agarose beads for 1–2 h at 4 °C. Thereafter, the software (https://www.combosyn.com/) based on the Chou–Talalay immunoprecipitates were collected by centrifugation, washed three times method [30]. CI < 1 indicates a synergistic effect, CI = 1 indicates an with IP lysis buffer, mixed with 50 µL of Laemmli’s SDS-buffer, denatured additive effect and CI > 1 indicates an antagonistic effect. CDI < 0.7 and then subjected to immunoblot analysis for BIM and MCL-1. Anti-rabbit indicates a significant synergistic effect. HRP-conjugated Fc fragment-specific secondary antibody (Cat. No. 111- 035-046, dilution 1:10000, Jackson ImmunoResearch, West Grove, PA) was used to detect immune complexes in immunoblotting. 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Nat Commun. 2020;11:1996. via downregulation of HER2/ERK1/2/MMP-9 signaling. Int J Biochem Cell Biol. 21. Soderquist RS, Crawford L, Liu E, Lu M, Agarwal A, Anderson GR, et al. Systematic 2015;58:1–16. mapping of BCL-2 gene dependencies in cancer reveals molecular determinants of BH3 mimetic sensitivity. Nat Commun. 2018;9:3513. 22. Thomas RL, Roberts DJ, Kubli DA, Lee Y, Quinsay MN, Owens JB, et al. Loss of ACKNOWLEDGEMENTS MCL-1 leads to impaired autophagy and rapid development of heart failure. This study was supported in part by US National Institutes of Health (NIH) grant R01 Genes Dev. 2013;27:1365–77. CA242003 (DZ & GZ), R01 CA241191 (DZ & GZ), R01 CA219836 (DZ), R01 CA260239 23. Hikita H, Takehara T, Shimizu S, Kodama T, Li W, Miyagi T, et al. Mcl-1 and Bcl-xL (DZ & WZ) and Florida Department of Health grant 8JK04 (FJK and MZ-K). cooperatively maintain integrity of hepatocytes in developing and adult murine liver. Hepatology 2009;50:1217–26. 24. Weeden CE, Ah-Cann C, Holik AZ, Pasquet J, Garnier JM, Merino D, et al. Dual AUTHOR CONTRIBUTIONS inhibition of BCL-XL and MCL-1 is required to induce tumour regression in lung SK conceived, designed, and supervised the study, performed most of the in vitro and squamous cell carcinomas sensitive to FGFR inhibition. Oncogene 2018;37:4475–88. in vivo experiments, analyzed and interpreted data, and wrote and revised the 25. George J, Lim JS, Jang SJ, Cun Y, Ozretić L, Kong G, et al. Comprehensive genomic manuscript; PK assisted in the GEM study; NC and CH designed, performed LX47 PDX profiles of small cell lung cancer. Nature 2015;524:47–53. study and analyzed the data DT, JW, VB, NH, YY, and UD performed some of the 26. Faber AC, Farago AF, Costa C, Dastur A, Gomez-Caraballo M, Robbins R, et al. experiments; PZ and XZ synthesized and purified DT2216 and prepared the vehicle and Assessment of ABT-263 activity across a cancer cell line collection leads to a formulated DT2216 for the studies; WZ shared resources, revised and commented on potent combination therapy for small-cell lung cancer. Proc Natl Acad Sci USA. the manuscript; LJ, provided some of the SCLC cell lines, revised and commented on the 2015;112:E1288–96. manuscript; GZ supervised the synthesis, purification, and formulation of DT2216, and 27. Elgendy M, Cirò M, Hosseini A, Weiszmann J, Mazzarella L, Ferrari E, et al. Combination revised the manuscript; RH revised and offered constructive criticism to help improve of hypoglycemia and metformin impairs tumor metabolic plasticity and growth by the manuscript; MZ-K and FJK provided some of the reagents including SCLC cell lines modulating the PP2A-GSK3β-MCL-1 Axis. Cancer Cell. 2019;35:798–815.e5. and GEM model, revised and commented on the manuscript; DZ co-conceived and co- 28. Faber AC, Coffee EM, Costa C, Dastur A, Ebi H, Hata AN, et al. mTOR inhibition supervised the study, interpreted some of the data, provided resources and revised the specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/ manuscript. All authors discussed the results and commented on the manuscript. BCL-XL inhibition by suppressing MCL-1. Cancer Discov. 2014;4:42–52. 29. Pétigny-Lechartier C, Duboc C, Jebahi A, Louis MH, Abeilard E, Denoyelle C, et al. The mTORC1/2 inhibitor AZD8055 strengthens the efficiency of the MEK inhibitor trametinib to reduce the Mcl-1/[Bim and Puma] ratio and to sensitize ovarian COMPETING INTERESTS carcinoma cells to ABT-737. Mol Cancer Ther. 2017;16:102–15. SK, DT, PZ, GZ, and DZ are inventors of two patent applications for use of BCL-X 30. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the com- PROTACs as senolytic and antitumor agents. RH, GZ, and DZ are co-founders of and bined effects of multiple drugs or enzyme inhibitors. Adv Enzym Regul. have equity in Dialectic Therapeutics, which develops BCL-X /2 PROTACs to treat 1984;22:27–55. cancer. 31. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol. 2019;20:175–93. 32. Lee Y-C, Wang L-J, Huang C-H, Shi Y-J, Chang L-S. ABT-263-induced MCL1 ADDITIONAL INFORMATION upregulation depends on autophagy-mediated 4EBP1 downregulation in human Supplementary information The online version contains supplementary material leukemia cells. Cancer Lett. 2018;432:191–204. available at https://doi.org/10.1038/s41420-022-01296-8. 33. Schaffer BE, Park KS, Yiu G, Conklin JF, Lin C, Burkhart DL, et al. Loss of p130 accelerates tumor development in a mouse model for human small-cell lung Correspondence and requests for materials should be addressed to Sajid Khan. carcinoma. Cancer Res. 2010;70:3877–83. 34. Kellish P, Shabashvili D, Rahman MM, Nawab A, Guijarro MV, Zhang M, et al. Reprints and permission information is available at http://www.nature.com/ Oncolytic virotherapy for small-cell lung cancer induces immune infiltration and reprints prolongs survival. J Clin Invest. 2019;129:2279–92. 35. Ullman-Culleré MH, Foltz CJ. Body condition scoring: a rapid and accurate Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims method for assessing health status in mice. Lab Anim Sci. 1999;49:319–23. in published maps and institutional affiliations. 36. Kolb R, De U, Khan S, Luo Y, Kim M-C, Yu H, et al. Proteolysis-targeting chimera against BCL-XL destroys tumor-infiltrating regulatory T cells. Nat Commun. 2021;12:1281. 37. Anderson GR, Wardell SE, Cakir M, Crawford L, Leeds JC, Nussbaum DP, et al. PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation. Sci Transl Med. 2016;8:369ra175. Open Access This article is licensed under a Creative Commons 38. Gardner EE, Connis N, Poirier JT, Cope L, Dobromilskaya I, Gallia GL, et al. Attribution 4.0 International License, which permits use, sharing, Rapamycin rescues ABT-737 efficacy in small cell lung cancer. Cancer Res. adaptation, distribution and reproduction in any medium or format, as long as you give 2014;74:2846–56. appropriate credit to the original author(s) and the source, provide a link to the Creative 39. Thummuri D, Khan S, Underwood PW, Zhang P, Wiegand J, Zhang X, et al. Commons license, and indicate if changes were made. The images or other third party Overcoming gemcitabine resistance in pancreatic cancer using the BCL-XL- material in this article are included in the article’s Creative Commons license, unless specific degrader DT2216. Mol Cancer Ther. 2022;21:184–92. indicated otherwise in a credit line to the material. If material is not included in the 40. Lee EF, Harris TJ, Tran S, Evangelista M, Arulananda S, John T, et al. BCL-XL and article’s Creative Commons license and your intended use is not permitted by statutory MCL-1 are the key BCL-2 family proteins in melanoma cell survival. Cell Death Dis. regulation or exceeds the permitted use, you will need to obtain permission directly 2019;10:342. from the copyright holder. To view a copy of this license, visit http:// 41. Del Gaizo Moore V, Letai A. BH3 profiling-measuring integrated function of the creativecommons.org/licenses/by/4.0/. mitochondrial apoptotic pathway to predict cell fate decisions. Cancer Lett. 2013;332:202–5. 42. Fraser C, Ryan J, Sarosiek K. BH3 profiling: a functional assay to measure apop- © The Author(s) 2023 totic priming and dependencies. Methods Mol Biol. 2019;1877:61–76. Cell Death Discovery (2023) 9:1 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice

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www.nature.com/cddiscovery ARTICLE OPEN Co-targeting BCL-X and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice 1,2,3✉ 4,5 6 3 3 7 7 Sajid Khan , Patrick Kellish , Nick Connis , Dinesh Thummuri , Janet Wiegand , Peiyi Zhang , Xuan Zhang , 3,8,9 3 1,3 10 11 10 7 Vivekananda Budamagunta , Nan Hua , Yang Yang , Umasankar De , Lingtao Jin , Weizhou Zhang , Guangrong Zheng , 12 6 4 13 1,2,3 Robert Hromas , Christine Hann , Maria Zajac-Kaye , Frederic J. Kaye and Daohong Zhou © The Author(s) 2023 Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-X and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on- target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-X degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co- dependent on BCL-X and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-X /MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-X degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-X and MCL-1. Cell Death Discovery (2023) 9:1 ; https://doi.org/10.1038/s41420-022-01296-8 INTRODUCTION thus an urgent and unmet need to find newer treatment Small-cell lung cancer (SCLC) is a difficult-to-treat subtype of strategies to effectively treat SCLC. pulmonary carcinomas with a 5-year survival rate of ~5% and SCLC has historically been treated as a homogeneous disease. median survival of less than a year [1, 2]. First-line treatment for However, it has been recently demonstrated that inter- and intra- SCLC with combinations of a platinum-based agent (cisplatin or tumoral heterogeneity occurs in SCLC, is primarily responsible for carboplatin) and etoposide (a topoisomerase-II inhibitor) has reduced therapeutic efficacy and resistance [6–9]. Since unique remained largely unchanged for almost 35 years. Recently, a triplet resistance mechanisms occur in response to specific therapies, of atezolizumab (a PD-L1 antibody), carboplatin, and etoposide specific oncoproteins and/or pathways need to be defined and was approved as first-line therapy for extensive-stage SCLC but targeted for effectively treating SCLC. Aberrant expression of anti- provides only a moderate (~2 months) increase in overall survival apoptotic BCL-2 family proteins has also been shown to account compared to chemotherapy alone [3, 4]. More importantly, the for intratumoral heterogeneity and therapeutic resistance in SCLC SCLC tumors are initially responsive to chemotherapy, however, [6, 10]. This has been therapeutically exploited with a dual BCL-X / relapse occurs in a majority (>80%) of cases, and there is no BCL-2 inhibitor ABT263 (navitoclax) that shows high efficacy in effective second-line treatment for relapsed SCLC [4, 5]. There is preclinical models of SCLC [11, 12]. Unfortunately, the single-agent 1 2 Department of Biochemistry & Structural Biology, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Mays Cancer Center, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Department of Pharmacodynamics, College of Pharmacy, University of Florida, 4 5 Gainesville, FL, USA. Department of Anatomy & Cell Biology, College of Medicine, University of Florida, Gainesville, FL, USA. Department of Pediatrics, College of Medicine, 6 7 University of Florida, Gainesville, FL, USA. Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA. Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, USA. Genetics and Genomics Graduate Program, Genetics Institute, College of Medicine, University of Florida, 9 10 Gainesville, FL, USA. Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA. Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA. Department of Molecular Medicine, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Department of Medicine, Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Division of Hematology and Oncology, Department of Medicine, College of Medicine, University of Florida, Gainesville, FL, USA. email: khans10@uthscsa.edu Received: 30 September 2022 Revised: 20 December 2022 Accepted: 22 December 2022 Official journal of CDDpress 1234567890();,: S. Khan et al. Fig. 1 The SCLC cell lines show differential survival dependence on BCL-2 family proteins. a The mRNA expression of BCL2 family anti- apoptotic genes as obtained from U Cologne study via cBioPortal. The mRNA expressions are depicted as Log2 (value + 1) from the RNA sequencing (RNA seq) dataset (n = 81 patient samples). b The EC values of A1155463 (a selective BCL-X inhibitor), venetoclax (a selective 50 L BCL-2 inhibitor), S63845 (a selective MCL-1 inhibitor), navitoclax (a BCL-X /BCL-2 dual inhibitor) and DT2216 (a selective BCL-X PROTAC L L degrader) were derived from % viability of indicated human SCLC cell lines after they were incubated with increasing concentrations of these inhibitors for 72 h. The data are presented from a single experiment performed in three replicate cell cultures. Similar results were obtained in two additional experiments performed with H146 and H378, and one additional independent experiment performed with H187, H211, and H209 cell lines. When an inhibitor showed an EC of ≤1 μM in any given cell line, the cell line was considered as sensitive to that inhibitor and dependent on its target BCL-2 family protein. efficacy of navitoclax against SCLC in phase-II clinical trials was limiting toxicities. We have devised a safer strategy to target these limited [13] because of the intratumoral heterogeneity and tumors as we found that BCL-X /MCL-1 co-dependent SCLC cells dependence of some of the cells within a tumor on other BCL-2 can be synergistically killed with a combination of DT2216 (a family proteins such as MCL-1. More importantly, the clinical use selective BCL-X PROTAC degrader) and AZD8055 (an mTOR of ABT263 is hampered by dose-limiting severe thrombocytopenia inhibitor). This was achieved by selective degradation of BCL-X caused by BCL-X inhibition [14, 15]. We are now able to safely and inhibition of MCL-1 expression in tumor cells with DT2216 and target BCL-X using proteolysis targeting chimeras (PROTACs, such AZD8055, respectively, therefore reducing on-target toxicity to as DT2216) without causing appreciable platelet toxicity (throm- platelets and other normal cells. In vivo, the combination of bocytopenia) as recently reported by our group [16–20]. DT2216 and AZD8055 strongly inhibited the growth of cell line- In this study, we aimed to develop a synergistic and safe derived and patient-derived xenograft (PDX) tumors and reduced therapeutic strategy for SCLC. This was achieved first by profiling tumor burden as well as increased survival of a murine Rb1/p53/ the heterogeneity of survival dependence of a panel of SCLC cell p130 SCLC genetically engineered mouse (GEM) model without lines on the BCL-2 family anti-apoptotic proteins by BH3 mimetic causing appreciable thrombocytopenia or other normal tissue screening, where we used BH3 mimetics to target specific BCL-2 injuries. anti-apoptotic proteins followed by measurement of cell viability [21]. We defined a subset of SCLC cell lines that are co-dependent on BCL-X and MCL-1 for survival and focused on testing the RESULTS ability to target these cell lines effectively and safely. Currently, BH3 mimetic screening identifies DT2216 sensitive and such tumors cannot be safely targeted with available inhibitors. resistant SCLC cells in vitro For example, MCL-1 inhibition causes severe cardiotoxicity, and We first explored the mRNA expression of anti-apoptotic BCL-2 co-targeting BCL-X and MCL-1 with commercially available family genes in patient tumors via cBioPortal [25]. These data inhibitors further exacerbates normal tissue injury and causes showed that BCL2, BCL2L1 (BCL-X coding gene), MCL1, and lethality [22–24]. Therefore, we need to develop an alternate BCL2A1 (BFL-1 coding gene) are abundantly expressed in most strategy to selectively suppress BCL-X and MCL-1 in co- SCLC patients’ tumors (Fig. 1a). Unfortunately, no healthy lung dependent SCLC tumors to avoid their on-target and dose- tissue transcriptomics data were available for a side-by-side Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 2 A subset of SCLC cell lines is co-dependent on BCL-X and MCL-1. a Viability graphs of representative SCLC cell lines showing dependence on individual or a combination of BCL-2 family anti-apoptotic proteins. The data are presented as mean ± SD from a single experiment (n = 3 replicate cell cultures). Similar results were obtained in one additional independent experiment performed with H209 and H211, and two additional independent experiments performed with H378 cells. b, c Viability of H378 and H1048 SCLC (b) and NHBE (c) cells after they were treated with increasing concentrations of DT2216 (DT) or S63845 (S) or their combination (1:1 ratio) for 72 h. EC and CI values are shown. The data are presented as mean ± SD from a representative experiment (n = 3 replicate cell cultures). Similar results were obtained in one additional independent experiment. comparison to show whether or not these genes were over- these may co-depend on a combination of either BCL-X /MCL-1 or expressed in SCLC. Therefore, to establish the importance of these BCL-2/MCL-1 or BCL-X /BCL-2/MCL-1 (Fig. 1b). These results BCL-2 family of anti-apoptotic proteins in SCLC, we systematically suggest that BCL-X is a key pro-survival protein in SCLC and evaluated the effects of inhibitors of BCL-2 family anti-apoptotic DT2216 might have a broad-spectrum antitumor activity than a proteins, i.e., A1155463 (a selective BCL-X inhibitor), venetoclax (a BCL-X inhibitor in killing BCL-X -dependent SCLC cells. L L L selective BCL-2 inhibitor), S63845 (a selective MCL-1 inhibitor) and navitoclax (a BCL-X /BCL-2 inhibitor) on the viability of a panel of A combination of MCL-1 inhibitor with BCL-X inhibitor or L L 20 SCLC cell lines, which was named as the BH3 mimetic screening PROTAC is not selective to tumor cells [21]. The cell lines with EC < 1 µM for a particular inhibitor were In the BH3 mimetic screening, SCLC cell lines showed varying considered sensitive. SCLC cell lines showed differential depen- responses to the inhibitors of different BCL-2 family anti-apoptotic dence on different BCL-2 family anti-apoptotic proteins, with a proteins. Based on these dependencies, we divided SCLC cell lines majority of them depending on BCL-X (9/20, 45%), while some into different categories; 1) primarily dependent on BCL-X (such L L depended on a combination of BCL-X and BCL-2 (3/20, 15%), and as H1963), 2) mainly dependent on BCL-2 (such as H889), 3) mostly only a few depended on BCL-2 (2/20, 10%) or MCL-1 (2/20, 10%), dependent on MCL-1 (such as H209), 4) BCL-X and BCL-2 co- as indicated by their sensitivity to these specific inhibitors (Fig. 1b). dependent (such as H1059), 5) BCL-X and MCL-1 co-dependent One cell line (H211) was found to be sensitive to all of these (such as H378), and 6) BCL-X , BCL-2, and MCL-1 dependent (such inhibitors indicating that all three, i.e., BCL-X , BCL-2, and MCL-1 as H211) (Fig. 2a). When two or more proteins were needed to be are important for its survival. These findings are corroborated by simultaneously inhibited in order to sensitize a particular cell line, the transcriptomic profiling suggesting that BCL-X is one of the we called it co-dependent on those proteins. For example, H1059 highly expressed BCL-2 family anti-apoptotic proteins, and there- cells were only sensitive upon simultaneous inhibition of BCL-X fore, a promising therapeutic target in SCLC tumors [10–12]. and BCL-2 using navitoclax, therefore, we categorized them as Further, we evaluated the activity of our BCL-X PROTAC degrader BCL-X and BCL-2 co-dependent. Similarly, H378 cells were L L DT2216 on the viability of these SCLC cell lines where 50% (10/20) sensitive to simultaneous inhibition of BCL-X and MCL-1, and cell lines were found to be sensitive to DT2216 with an EC of therefore, categorized as BCL-X /MCL-1 co-dependent. These 50 L <1 µM. All of the cell lines that were found to be sensitive to the results indicate that SCLC cells are heterogeneous in their BCL-X inhibitor were sensitive to DT2216 as well. Interestingly, the dependencies on BCL-2 anti-apoptotic proteins. H1059 cell line was not sensitive to the BCL-X inhibitor but was Next, we focused on targeting BCL-X and MCL-1 co-dependent L L found to be sensitive to DT2216. Six of the cell lines did not cells, as these types of SCLC tumors are highly resistant to respond to any of these inhibitors or DT2216 suggesting that chemotherapy and cannot be safely eradicated with the currently Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 3 mTOR inhibitor AZD8055 selectively suppresses MCL-1 expression in tumor cells. a, b Immunoblot analyses of MCL-1, BCL-X and L, BCL-2 in SCLC H378 and H1048 cell lines (a) and NHBE cells (b) after they were treated with indicated agents (1 µM each) for 24 h. Normalized densitometric values for MCL-1 blots are shown underneath. c, d Immunoblot analysis of mTOR substrates (p-4EBP1 and p-S6), MCL-1, BCL-X , and BCL-2 after 24 h treatment with indicated concentrations of AZD8055 in H378 and H1048 SCLC cell lines (c) and NHBE cells (d). The uncropped immunoblot images related to this figure are provided in the “Supplemental Material” file. available inhibitors of BCL-2 family proteins because the inhibitor that primarily targets CDK9), AZD8055 (an ATP- combination of BCL-X and MCL-1 inhibitors causes severe normal competitive catalytic mTORC1/2 inhibitor), TD-19 (a cancerous tissue toxicities and lethality in mice [23, 24, 26]. This was inhibitor of protein phosphatase 2A inhibitor) and piperazine (a confirmed by our study showing that a combination of DT2216 or protein phosphatase 2A [PP2A] activator). All of these therapeutic A1155463 with MCL-1 inhibitor (S63845) synergistically kills not agents have previously been shown to suppress MCL-1 expression only tumor cells (Fig. 2b), but also normal cells from different through different mechanisms [10, 26–29]. Here, we aimed to tissue origins such as human bronchial epithelial cells (NHBE), determine whether any of these agents could selectively suppress colon fibroblasts (CCD-18Co) and lung fibroblasts (WI38) (Fig. 2c; MCL-1 expression in tumor cells while having minimal/no supplementary Fig. 1a). Of note, S63845 treatment alone was also significant effect on the expression of MCL-1 in normal cells. In highly toxic to NHBE cells, which indicates that these cells agreement with previous reports, doxorubicin, SNS-032 and primarily depend on MCL-1 for survival. These findings do not AZD8055 caused significant suppression of MCL-1 expression in support using the combination of a BCL-X inhibitor/PROTAC and all tested SCLC cell lines (Fig. 3a; Supplementary Fig. 1b). Among an MCL-1 inhibitor to treat BCL-X and MCL-1 co-dependent SCLC these inhibitors tested, only AZD8055 showed minimal effect on in the clinic. the expression of MCL-1 in normal cells (Fig. 3b; Supplementary Fig. 1c). None of these agents, except SNS-032, significantly altered DT2216+AZD8055 combination synergistically kills BCL-X / BCL-X or BCL-2 levels in both tumor cells as well as normal cells. L L MCL-1 co-dependent SCLC cells through degradation of BCL- Further, we analyzed the dose-dependent effect of AZD8055 on X and suppression of MCL-1 expression, respectively, in a the expression of MCL-1, BCL-X and BCL-2 in H378 and H1048 L L, tumor cell-selective manner SCLC and NHBE normal cells. AZD8055 caused dose-dependent Next, we sought to identify a strategy to selectively suppress suppression of MCL-1 expression in tumor cells without any MCL-1 expression in SCLC cells by rationally targeting certain observable effect in normal cells. As expected, AZD8055 had no tumorigenic proteins/pathways. We screened several clinical-stage observable effects on the expression of BCL-X and BCL-2 in all the compounds and FDA-approved drugs including doxorubicin (a tested cell lines. Of note, BCL-2 expression was not detected in topoisomerase II inhibitor and a commonly used chemotherapeu- NHBE cells via immunoblotting. Also, AZD8055 dose-dependently tic), SNS-032 (a promiscuous cyclin-dependent kinase [CDK] inhibited activation of the mTOR downstream targets, i.e., p-4EBP1 Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 4 DT2216+AZD8055 combination synergistically kills BCL-X /MCL-1 co-dependent SCLC cells through disruption of MCL-1/BIM interaction. a, b Viability of H378 and H1048 SCLC (a) and NHBE (b) cells after they were treated with increasing concentrations of DT2216 (DT) or AZD8055 (AZD) or their combination (1:1 ratio) for 6 days. EC and CI values are shown. The data presented in a and b are mean ± SD from a representative experiment (n = 3 replicate cell cultures). Similar results were obtained in two additional independent experiments. c, d Immunoblot analyses of full-length PARP (fPARP) and cleaved PARP (cPARP) in SCLC H378, H1048 (c), and NHBE (d) cell lines after they were treated with 1 µM each of DT2216 or AZD8055 or their combination for 24 h. e Immunoprecipitation analysis of MCL-1 followed by immunoblotting of BIM and MCL-1 in H1048 cells after they were treated with 1 µM each of DT2216 and/or AZD8055 for 24 h. Immunoblot analyses of BIM, MCL-1, and BCL-X in input samples are shown on the right. The uncropped immunoblot images related to this figure are provided in the “Supplemental Material” file. and p-S6, in both SCLC and NHBE cells indicating a desired on- inhibitor. Everolimus is FDA-approved for use as an immunosup- target effect (Fig. 3c, d). pressant and for treating certain tumors. We wondered whether Since AZD8055 was found to selectively suppress MCL-1 everolimus can also synergistically kill BCL-X /MCL-1 co- expression in SCLC cells, we next evaluated it in combination dependent SCLC cells when combined with DT2216. Indeed, the with DT2216 on the viability of BCL-X /MCL-1 co-dependent SCLC combination of everolimus and DT2216 synergistically reduced cells as well as normal cells. Out of 11 SCLC cell lines that were the viability of H378 and H1048 cells, as well as more effectively resistant to BCL-X inhibitor (A1155463) alone in Fig. 1b, induced apoptosis compared to individual agents in H1048 cells we selected the five most resistant cell lines to test against (Supplementary Fig. 4a, b). However, unlike AZD8055, everolimus the combination of BCL-X inhibitor and MCL-1 inhibitor. Out of alone was less effective in inhibiting the viability of H1048 cells, these five, three cell lines (H378, H1048, and DMS53) were found which might be because it caused even lesser apoptosis to be sensitive to dual BCL-X and MCL-1 inhibition. Further, we compared to AZD8055 (Supplementary Fig. 3b, 4b). This was used the two most sensitive (H378 and H1048) cell lines to test the accompanied by a significant reduction in MCL-1 expression in combination of DT2216 and AZD8055. The DT2216 + AZD8055 H1048 cells after everolimus treatment (Supplementary Fig. 4c). combination was found to synergistically inhibit the viability of These results suggest that the tumor-specific inhibition of only H378 and H1048 SCLC cells, but not normal NHBE and CCD-18Co mTORC1 is sufficient for the suppression of MCL-1 expression. cells, as assessed by CI values at EC (concentration with 75% Also, mTOR seems to differentially regulate MCL-1 in tumor and viability loss) and EC (concentration with 90% viability loss) normal cells, therefore we could not see MCL-1 downregulation using Chou-Talalay method [30] (Fig. 4a, b; Supplementary Fig. even after efficient mTOR inhibition in NHBE cells. 1d). Notably, AZD8055 treatment alone led to partial inhibition of To gain further insights into the mechanism of synergistic activity H1048, NHBE, and CCD-18Co cell viability which mainly attributed of DT2216+ AZD8055, we performed a co-immunoprecipitation to its antiproliferative effects. Importantly, the combination of (co-IP) analysis of MCL-1 and BIM because BIM acts as a BH3-only DT2216 and AZD8055 caused a complete loss of viability of tumor pro-apoptotic protein that can trigger apoptosis by activating the cells, but not normal cells. Therefore, the combination effects were apoptotic effectors BAX and BAK and the sequestration of BIM by synergetic in tumor cells, but not in normal cells, as indicated by MCL-1 or BCL-X inhibits apoptosis [31]. Since BIM is known to bind the CI values. DT2216 treatment induced a dose-dependent BCL- both BCL-X and MCL-1, we hypothesized that the degradation of X degradation in H1048 cells as expected (Supplementary Fig. 2). BCL-X with DT2216 may lead to an increased association of BIM L L Since 1 µM of DT2216 was found to completely deplete BCL-X ,we with MCL-1, and co-treatment with DT2216+ AZD8055 may disrupt selected this concentration for further in vitro experiments. In this interaction. In line with our hypothesis, we found that DT2216 addition, we found that the combination of DT2216 and AZD8055 treatment increases the BIM/MCL-1 association which was disrupted was more effective compared to individual agents in inhibiting when the cells were treated with the DT2216+ AZD8055 combina- H1048 cell survival and inducing apoptosis by performing the tion (Fig. 4e). Interestingly, increased BIM binding seems to stabilize long-term clonogenic assay and flow cytometric analysis, respec- MCL-1 upon DT2216 treatment, and the MCL-1 levels are tively (Supplementary Fig. 3a, b). Furthermore, the suppressed in the combination-treated cells. Since BCL-X inhibition DT2216 + AZD8055 combination profoundly induced PARP clea- has been shown to increase MCL-1 levels in some cancer cell lines as vage compared to individual agents in tumor cells, but not in acompensatorymechanism [32]. So, the increased MCL-1 levels NHBE cells, which again confirmed the tumor cell-selectivity of the upon DT2216 treatment may be due to either BIM-induced combination (Fig. 4c, d). stabilization and/or a compensatory mechanism that needs further Unlike AZD8055 which inhibits both mTORC1 and mTORC2, exploration. everolimus is a rapamycin derivative and a selective mTORC1 Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 5 The combination of DT2216 and AZD8055 has stronger antitumor responses in H1048 xenograft and SCLC PDX models. a, b Tumor volume (a) and mouse body weight (b) change in H1048 xenografts after treatment with vehicle (Veh), DT2216 (DT, 15 mg/kg/q4d, i.p.), AZD8055 (AZD, 16 mg/kg/5x week, p.o.) or a combination of DT and AZD. Data are presented as mean ± SEM (n = 5 mice in Veh and single- treatment groups, and six mice in combination group at the start of treatment). c Platelet counts 24 h after the first dose of DT2216 (DT, 15 mg/kg/q4d, i.p.) and/or AZD8055 (AZD, 16 mg/kg/5x week, p.o.) in H1048 xenograft mice from a separate experiment (n = 7 mice in Veh and single-treatment groups, and eight mice in combination group). d Immunoblot analysis of indicated proteins in H1048 xenograft tumors at the end of treatments as in a (n = 3 mice per group). e Densitometry of immunoblots shown in panel d (mean ± SEM). f, g Tumor volume (f) and mouse body weight (g) changes in LX47 PDX tumors after treatment with veh, DT2216 (DT, 15 mg/kg/q4d, i.p.) or AZD8055 (AZD, 16 mg/ kg/5x week, p.o.) or a combination of DT and AZD. Data are presented as mean ± SEM (n = 8 mice in single-treatment groups, and nine mice in Veh and combination groups at the start of treatment). Statistical significance in a, c, e, and f was determined by a two-sided unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. The uncropped immunoblot images related to this figure are provided in the “Supplemental Material” file. DT2216+AZD8055 combination synergistically inhibits tumor be clinically safe [16]. Therefore, a 25–30% reduction in total platelet growth in SCLC xenograft and PDX models count may likely not be of any clinical concern. Next, we confirmed To further assess the combined mTOR inhibition and BCL-X that DT2216 and AZD8055 led to a significant reductioninBCL-X L L degradation as a potential therapeutic strategy for BCL-X and and MCL-1 levels, respectively, which was associated with significant MCL-1 co-dependent SCLC, we first investigated the efficacy of inhibition of mTOR substrates (p-4EBP1 and p-S6) upon AZD8055 DT2216+ AZD8055 combination using the H1048 xenograft tumor treatment in H1048 tumor lysates. Interestingly, the combination model. In agreement with the in vitro results, DT2216 alone had no also caused a significant decrease in BCL-2 protein levels in these effect on H1048 tumor growth. Although AZD8055 alone showed tumor samples (Fig. 5d, e). some efficacy, the effect was not significant. On the other hand, the Since the PDX models better recapitulate human cancers in vivo, combination of DT2216 and AZD8055 led to significant inhibition of we next evaluated the effect of the combination of tumor growth (Fig. 5a). More importantly, the combination DT2216+ AZD8055 in the LX47-SCLC PDX model. We found that treatment appeared to be safe as indicated by no significant the combination significantly inhibited tumor growth in this PDX change in mouse body weights after the treatment (Fig. 5b). In model of SCLC, whereas single agents failed to have significant addition, we did not observe any gross organ toxicities upon effects(Fig. 5f). Again, we did not observe any significant body necropsy in mice treated with DT2216+ AZD8055. Furthermore, the weight loss with the combination treatment in these mice (Fig. 5g). combination caused a similar reduction (25–30%) in platelets as DT2216 alone, where platelet counts remained well above 2 × 10 DT2216+AZD8055 combination inhibits lung tumor growth per µL of blood (Fig. 5c). Notably, platelet counts above 2 × 10 per and increases mice survival in the Rb1/p53/p130 GEM model µL of blood in mice (equivalent to 5 × 10 per µL in humans) are not Next, we used the conditional-mutant Rb1/p53/p130 mouse model associated with a significant risk of hemorrhage and considered to of SCLC to test the efficacy of the DT2216+ AZD8055 combination. Cell Death Discovery (2023) 9:1 S. Khan et al. Fig. 6 The combination of DT2216 and AZD8055 has stronger antitumor responses as compared to individual agents in a GEM model of SCLC. a Experimental design of Rb1/p53/p130 GEM model. Mice were administered with 2.5 × 10 pfu/mouse of Ad5CMVCre-eGFP through nasal inhalation. Treatment was started 110 days post-infection and continued until day 150. Specifically, the mice were treated with vehicle (veh), AZD8055 (AZD, 16 mg/kg/5x week, p.o.) and AZD (16 mg/kg/5x week, p.o.) + DT2216 (DT, 15 mg/kg/q4d, i.p.) (n= 8, 8 and 7 mice in veh, AZD and combo groups, respectively). The figure was adapted from BioRender. b Kaplan–Meier survival analysis of mice as treated in a. The median survival time is shown on the right. At the end of the experiment (day 166), 2, 2, and 4 mice were alive in veh, AZD and DT + AZD groups, respectively. c Bouin’s-stained represented images of excised lungs from veh, AZD, and DT + AZD groups. The tumor nodules in the lungs are circled. As shown, the tumor nodules were considerably smaller in the lungs of combination-treated mice as compared to veh or AZD-treated mice. d The Number of tumor nodules was counted on both dorsal and ventral sides of the lungs and the average number of tumor nodules per mouse lungs in each group is shown (mean ± SEM, n = 7). Statistical significance was determined by a two-sided unpaired Student’s t test. **p < 0.01. e Hematoxylin and eosin (H&E) staining of representative lung sections in veh, DT, AZD, and DT + AZD groups from a separate experiment in which the mice were treated with veh, DT (15 mg/kg/q4d, i.p.), AZD (16 mg/kg/5x week, p.o.), and a combination of DT and AZD. The mice were euthanized 24 h after the last treatment. In these mice, Rb1, Tp53, and p130 tumor suppressor genes have mTOR inhibition in part via depleting tumor-infiltrating regulatory flanking lox sequences that are under the control of Cre T cells (TI-Tregs) as shown in our recent study [36]. Depletion of recombinase. These genes are deleted upon administration of TI-Tregs by DT2216 may stimulate antitumor immunity and Adenovirus (Ad5CMVCre-eGFP), specifically in the lung epithelial attenuate mTOR inhibition-induced immunosuppression and cells, and lead to tumor formation in the lungs [33, 34]. Since the spleen size reduction. Furthermore, we did not see any clinically Rb1/p53/p130 mouse tumors closely resemble human SCLC, it is a significant reduction in platelets and other blood cell counts in the suitable preclinical mouse model to evaluate newer therapeutics treatment cohorts (Supplementary Fig. 5b, c). Overall, these results against SCLC. We determined a mean tumor onset time of suggest that the DT2216+ AZ8055 combination may be a 110 days after administration of adenovirus titer of 2.5 × 10 pfu/ promising therapeutic strategy, particularly against BCL-X /MCL-1 mouse to induce the formation of small visible tumor nodules in co-dependent SCLC, and therefore this combination could the mouse lungs. Therefore, we started treating the mice after potentially be tested in clinical trials in the future. 110 days with DT2216 and/or AZD8055 for up to 150 days (Fig. 6a). We did not include the DT2216 alone group because DT2216 was not found to have any significant effect on tumor growth when DISCUSSION used alone in this model in a previous study as shown in Fig. 6e. SCLC shows high inter- and intra-tumoral heterogeneity which is Mice were euthanized, and the survival was recorded at the primarily responsible for the therapeutic resistance [6–9]. This humane endpoint when the body condition score of animals heterogeneity is in part attributed to an aberrant expression of declined [35]. At the end of the experiment i.e, 150 days after anti-apoptotic BCL-2 family proteins which are crucial for the treatment, 2/8 (25%), 2/8 (25%) and 4/7 (~57%) mice were alive in survival of SCLC cells [10–12]. In the present study, we first the vehicle, AZD8055, and combination-treatment groups, respec- established the survival dependence of SCLC cell lines on different tively. The median survival time was 131, 149.5, and >166 days in BCL-2 family anti-apoptotic proteins using BH3 mimetic screening. the vehicle, AZD8055, and the combination-treatment group, We found that most SCLC cell lines are dependent on BCL-X for respectively (Fig. 6b). We excised the lungs from these mice and their survival and are highly sensitive to a BCL-X inhibitor or our observed that the combination-treated lungs had much smaller recently developed BCL-X PROTAC DT2216 (now a clinical tumor nodules as compared to vehicle or AZD8055 treatments candidate in the phase-I trial) [16, 17]. On the other hand, some (Fig. 6c). Moreover, the number of total tumor nodules per lungs SCLC cell lines were found to be dependent on multiple co- was also significantly reduced in the combination-treated mice expressed BCL-2 family proteins such as concurrent BCL-X and (Fig. 6d). The histopathological staining in the lungs from a BCL-2, or concurrent BCL-X and MCL-1. BCL-X /BCL-2 co- L L separate experiment further evidenced the reduction in tumor dependent cell lines could be targeted with navitoclax, however, burden in the combination-treated mice (Fig. 6e). We did not see the clinical translation of navitoclax is hampered by dose-limiting any significant changes in the lungs, spleen, liver, or kidney on-target toxicity of thrombocytopenia [14, 15]. More importantly, weights after combination treatment (Supplementary Fig. 5a). The BCL-X /MCL-1 co-dependent tumor cells cannot be safely targeted reductions in spleen weight in AZD-treated mice might be with the currently available inhibitors because the combination of attributable to the immunosuppressive effect of mTOR inhibitors. BCL-X and MCL-1 inhibitors causes severe tissue damage and DT2216 might be able to reduce the immunosuppressive effect of lethality in mice [22–24]. Therefore, finding a strategy to Cell Death Discovery (2023) 9:1 S. Khan et al. selectively target BCL-X and MCL-1 in tumors without causing YAP1 [5, 7]. Each of these molecular subtypes shows distinct significant on-target toxicity was the primary goal of our current therapeutic vulnerabilities [8, 9]. For example, ASCL1-high subtype study for treating an important difficult-to-treat subset of SCLC. (classic or neuroendocrine (NE) SCLC) [7] has been shown to be Since systemic MCL-1 inhibition with currently available sensitive to BCL-2 inhibition with ABT263 [43]. Interestingly, we inhibitors leads to normal tissue toxicity and lethality when observed that most variant-SCLC cells (non-NE consisting of combined with a BCL-X inhibitor/PROTAC, we sought to identify a POU2F3 and/or YAP1 subtype) [7] were dependent on the co- strategy to selectively suppress MCL-1 expression in SCLC cells. expression of BCL-X and MCL-1. Although these subtypes are not After screening several compounds that target oncogenic path- being used to direct treatment decisions for SCLC at present, we ways known to upregulate MCL-1 [10, 26–29], we identified that expect that POU2F3 and/or YAP1 SCLC subtypes could be better AZD8055, a mTORC1/2 ATP-competitive inhibitor, and everolimus, targeted with a combination of DT2216 and an mTOR inhibitor. In a selective mTOR1 inhibitor, can selectively suppress MCL-1 this case, immunohistochemical or proteomics profiling of four of expression in SCLC cells but not in normal cells. In contrast, other these transcriptional regulators could be a better alternative to compounds such as doxorubicin and SNS-032 non-specifically BH3 profiling for stratifying patients who can specifically benefit downregulated MCL-1 in tumor cells as well as in normal cells. This from the DT2216 + mTOR inhibitor combination. In the current was correlated with high basal mTOR activation in MCL-1- study, we used AZD8055 as a proof of concept, but it would be dependent SCLC cell lines (data not shown). The mTOR activation suitable to use an FDA-approved mTOR inhibitor (such as has been shown to enhance cap-dependent MCL-1 translation, everolimus) in combination with DT2216 in clinical trials. and therefore, inhibition of mTOR suppresses MCL-1 at post- In conclusion, the findings from our in vitro and in vivo studies translational level [26, 28, 29, 37]. Of note, the combination of suggest that the combination of DT2216 + AZD8055 is synergistic DT2216 with AZD8055 or everolimus synergistically inhibited the and somewhat tumor-selective against the BCL-X /MCL-1 co- viability and induced apoptosis in BCL-X and MCL-1 co- dependent subset of SCLC and is tolerable in mice without dependent SCLC cell lines, but not the normal cells from different appreciable on-target toxicity and normal tissue injury. These tissue origins including lungs and colon. findings may have a high potential for near-term clinical Our further investigation using different in vivo SCLC models translation as the combination of DT2216 and an FDA-approved including conventional xenografts, PDX, and Rb1/p53/p130 GEM mTOR inhibitor (such as everolimus) can be rapidly evaluated in model also suggests that the combination of DT2216 and SCLC patients, given that DT2216 is already in the phase-I clinical AZD8055 can effectively inhibit the SCLC growth in mice. More trial (Identifier: NCT04886622). This kind of approach can help to importantly, the combination appeared to be safe as evidenced by effectively treat an important and difficult-to-treat subset of SCLC no significant decrease in mouse body weights as well as no patients in the near future. clinically significant reduction in different blood cells including platelets, and the absence of any observable tissue pathology. Interestingly, results using the SCLC GEM model, which closely MATERIALS AND METHODS recapitulates genetic heterogeneity found in human SCLC, Cell lines and culture All the SCLC cell lines, except DMS53 and DMS114, were obtained from the indicate that the combination of DT2216 + AZD8055 strongly original NCI-Navy Medical Oncology source supply [44]. DMS53 and inhibits tumor growth in the lungs and increased the survival of DMS114 SCLC cell lines, CCD-18Co normal colon fibroblasts, and WI-38 mice. The effectiveness of the combination in the GEM model was lung fibroblasts were purchased from the American Type Culture encouraging because the tumors in this model have been shown Collection (ATCC, Manassas, VA). SCLC cell lines were cultured in RPMI- to be highly resistant to cisplatin plus etoposide doublet 1640 medium (Cat. No. 22400–089, Thermo Fisher, Waltham, MA). CCD- chemotherapy [26]. 18Co and WI-38 cells were cultured in Dulbecco’s modified Eagle’s medium These findings have important clinical implications because (DMEM) (Cat. No. 12430-062, Thermo Fisher). The culture media were tumor-specific targeting of BCL-X and MCL-1 has high therapeutic supplemented with 10% heat-inactivated fetal bovine serum (FBS, Cat. No. value. Two prior studies have found that a combination of S11150H, Atlanta Biologicals, GA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pen-Strep, Cat. No. 15140122, Thermo Fisher). NHBE cells navitoclax and AZD8055 synergistically kills different tumor cells were purchased from Lonza (Cat. No. CC-2541, Basel, Switzerland), and from SCLC and BRAF/KRAS-mutated colorectal cancer [26, 28]. were cultured in Lonza’s bronchial epithelial cell growth medium (Cat. No. Moreover, a study from Dr. Christine Hann’s group has demon- CC-3170) with supplements and growth factors (CC-4175). The stocks of strated synergy between rapamycin and ABT737 (a predecessor NCI SCLC cell lines that we used were STR profiled by NIH, so the compound of navitoclax) against different PDX models of SCLC authenticity of the cell lines remained preserved. All cultures were [38]. In another study, the combined inhibition of BCL-X and confirmed for Mycoplasma negativity using the MycoAlert Mycoplasma mTOR was found to synergistically induce apoptosis in PIK3CA- Detection Kit (Cat. No. LT07–318). All the cell lines were maintained in a mutated breast cancer [37]. However, these studies did not aim to humidified incubator at 37 °C and 5% CO . comprehensively evaluate tumor-selectivity and on-target toxi- cities of this combination (especially in mouse models). Given that Chemical compounds BCL-X inhibitors including navitoclax cause thrombocytopenia, a DT2216 was synthesized in Dr. Guangrong Zheng’s laboratory (University of combination of navitoclax with AZD8055 (or another mTOR Florida, Gainesville, FL) according to the previously described protocol [16]. inhibitor) may not be clinically feasible [14, 15]. In contrast, by AZD8055 (Cat. No. HY-10422) and everolimus (Cat. No. HY-10218) were combining AZD8055 with platelet-sparing BCL-X degrader purchased from MedChemExpress (Monmouth Junction, NJ). A1155463 (Cat. No. S7800), ABT199 (Cat. No. S8048), S63845 (Cat. No. S8383), and DT2216, we achieved tumor-selective targeting of both MCL-1 ABT263 (Cat. No. S1001) were purchased from SelleckChem (Houston, TX). and BCL-X , respectively, without significant on-target toxicity both in cell culture and in mice. This approach can also be applied to other tumor types which are co-dependent on MCL-1 and BCL- Cell viability assays X [39, 40]. One limitation of our in vitro BH3 mimetic screening is The cell viability was measured by MTS assay according to the manufacturer’s protocol (Cat. No. G-111, Promega, Madison, WI) and as that it requires live cells, so it cannot be used to stratify patients described previously [16, 39]. EC values were determined using who will benefit from the combination. In that case, a dynamic GraphPad Prism software (GraphPad Software, La Jolla, CA). BH3 profiling assay would be more useful as demonstrated previously [41, 42]. More recently, SCLC tumors have been classified into four BH3 mimetic screening The screening was performed to determine the survival dependence of molecular subtypes based on the relative expression of four SCLC cells on different BCL-2 family anti-apoptotic proteins [21]. The cells transcriptional regulators, i.e., ASCL1, NEUROD1, POU2F3, and Cell Death Discovery (2023) 9:1 S. Khan et al. were treated in 96-well plates with either a specific inhibitor of BCL-2 were treated with vehicle, AZD8055 (16 mg/kg, 5 days a week, p.o.), and a family anti-apoptotic proteins such as A1155463 (selective BCL-X combination of DT2216 (15 mg/kg, q4d, i.p.) and AZD8055 (n = 7–8 mice inhibitor), venetoclax (selective BCL-2 inhibitor), and S63845 (selective per group). Survival events were scored when the body condition score (as MCL-1 inhibitor), or a non-specific inhibitor such as navitoclax (BCL-X /BCL- defined by the American Association for Laboratory Animal Science) of 2 dual inhibitor). Thereafter, the viability was assessed using the MTS assay animals declined or per absolute survival events [35]. The lungs were as described above. excised upon euthanizing the individual mice, stained in Bouins’ solution, and photographed as described previously [45, 46]. The number of visible tumor nodules was counted on the surface of each pair of lungs and is Co-immunoprecipitation presented as the average number of tumor nodules/lungs. Cell pellets were lysed in the Pierce IP lysis buffer (Cat. No. 87787; Thermo Fisher) supplemented with protease and phosphatase inhibitors as described previously [16, 39]. The supernatants were collected and Statistical analysis precleared by incubating with 1 µg of mouse anti-IgG (Cat. No. sc-2025; All the graphs presented in this manuscript were made and statistical Santa Cruz Biotechnology [SCB], Dallas, TX) and 20 µL of protein A/G-PLUS analyses were performed using the GraphPad Prism-9 software. A two- agarose beads (Cat. No. sc-2003; SCB) for 30 min at 4 °C. The supernatants sided unpaired Student’s t test was used for comparisons between the containing 1 mg of protein were incubated with 2 µg of anti-MCL-1 (Cat. means of the two groups. P < 0.05 was considered to be statistically No. sc-12756; SCB) or anti-IgG antibody overnight followed by incubation significant. The combination index (CI) was calculated using Compusyn with 25 µL protein A/G agarose beads for 1–2 h at 4 °C. Thereafter, the software (https://www.combosyn.com/) based on the Chou–Talalay immunoprecipitates were collected by centrifugation, washed three times method [30]. CI < 1 indicates a synergistic effect, CI = 1 indicates an with IP lysis buffer, mixed with 50 µL of Laemmli’s SDS-buffer, denatured additive effect and CI > 1 indicates an antagonistic effect. CDI < 0.7 and then subjected to immunoblot analysis for BIM and MCL-1. Anti-rabbit indicates a significant synergistic effect. HRP-conjugated Fc fragment-specific secondary antibody (Cat. No. 111- 035-046, dilution 1:10000, Jackson ImmunoResearch, West Grove, PA) was used to detect immune complexes in immunoblotting. 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Nat Commun. 2020;11:1996. via downregulation of HER2/ERK1/2/MMP-9 signaling. Int J Biochem Cell Biol. 21. Soderquist RS, Crawford L, Liu E, Lu M, Agarwal A, Anderson GR, et al. Systematic 2015;58:1–16. mapping of BCL-2 gene dependencies in cancer reveals molecular determinants of BH3 mimetic sensitivity. Nat Commun. 2018;9:3513. 22. Thomas RL, Roberts DJ, Kubli DA, Lee Y, Quinsay MN, Owens JB, et al. Loss of ACKNOWLEDGEMENTS MCL-1 leads to impaired autophagy and rapid development of heart failure. This study was supported in part by US National Institutes of Health (NIH) grant R01 Genes Dev. 2013;27:1365–77. CA242003 (DZ & GZ), R01 CA241191 (DZ & GZ), R01 CA219836 (DZ), R01 CA260239 23. Hikita H, Takehara T, Shimizu S, Kodama T, Li W, Miyagi T, et al. Mcl-1 and Bcl-xL (DZ & WZ) and Florida Department of Health grant 8JK04 (FJK and MZ-K). cooperatively maintain integrity of hepatocytes in developing and adult murine liver. Hepatology 2009;50:1217–26. 24. Weeden CE, Ah-Cann C, Holik AZ, Pasquet J, Garnier JM, Merino D, et al. Dual AUTHOR CONTRIBUTIONS inhibition of BCL-XL and MCL-1 is required to induce tumour regression in lung SK conceived, designed, and supervised the study, performed most of the in vitro and squamous cell carcinomas sensitive to FGFR inhibition. Oncogene 2018;37:4475–88. in vivo experiments, analyzed and interpreted data, and wrote and revised the 25. George J, Lim JS, Jang SJ, Cun Y, Ozretić L, Kong G, et al. Comprehensive genomic manuscript; PK assisted in the GEM study; NC and CH designed, performed LX47 PDX profiles of small cell lung cancer. Nature 2015;524:47–53. study and analyzed the data DT, JW, VB, NH, YY, and UD performed some of the 26. Faber AC, Farago AF, Costa C, Dastur A, Gomez-Caraballo M, Robbins R, et al. experiments; PZ and XZ synthesized and purified DT2216 and prepared the vehicle and Assessment of ABT-263 activity across a cancer cell line collection leads to a formulated DT2216 for the studies; WZ shared resources, revised and commented on potent combination therapy for small-cell lung cancer. Proc Natl Acad Sci USA. the manuscript; LJ, provided some of the SCLC cell lines, revised and commented on the 2015;112:E1288–96. manuscript; GZ supervised the synthesis, purification, and formulation of DT2216, and 27. Elgendy M, Cirò M, Hosseini A, Weiszmann J, Mazzarella L, Ferrari E, et al. Combination revised the manuscript; RH revised and offered constructive criticism to help improve of hypoglycemia and metformin impairs tumor metabolic plasticity and growth by the manuscript; MZ-K and FJK provided some of the reagents including SCLC cell lines modulating the PP2A-GSK3β-MCL-1 Axis. Cancer Cell. 2019;35:798–815.e5. and GEM model, revised and commented on the manuscript; DZ co-conceived and co- 28. Faber AC, Coffee EM, Costa C, Dastur A, Ebi H, Hata AN, et al. mTOR inhibition supervised the study, interpreted some of the data, provided resources and revised the specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/ manuscript. All authors discussed the results and commented on the manuscript. BCL-XL inhibition by suppressing MCL-1. Cancer Discov. 2014;4:42–52. 29. Pétigny-Lechartier C, Duboc C, Jebahi A, Louis MH, Abeilard E, Denoyelle C, et al. The mTORC1/2 inhibitor AZD8055 strengthens the efficiency of the MEK inhibitor trametinib to reduce the Mcl-1/[Bim and Puma] ratio and to sensitize ovarian COMPETING INTERESTS carcinoma cells to ABT-737. Mol Cancer Ther. 2017;16:102–15. SK, DT, PZ, GZ, and DZ are inventors of two patent applications for use of BCL-X 30. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the com- PROTACs as senolytic and antitumor agents. RH, GZ, and DZ are co-founders of and bined effects of multiple drugs or enzyme inhibitors. Adv Enzym Regul. have equity in Dialectic Therapeutics, which develops BCL-X /2 PROTACs to treat 1984;22:27–55. cancer. 31. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol. 2019;20:175–93. 32. Lee Y-C, Wang L-J, Huang C-H, Shi Y-J, Chang L-S. ABT-263-induced MCL1 ADDITIONAL INFORMATION upregulation depends on autophagy-mediated 4EBP1 downregulation in human Supplementary information The online version contains supplementary material leukemia cells. Cancer Lett. 2018;432:191–204. available at https://doi.org/10.1038/s41420-022-01296-8. 33. Schaffer BE, Park KS, Yiu G, Conklin JF, Lin C, Burkhart DL, et al. Loss of p130 accelerates tumor development in a mouse model for human small-cell lung Correspondence and requests for materials should be addressed to Sajid Khan. carcinoma. Cancer Res. 2010;70:3877–83. 34. Kellish P, Shabashvili D, Rahman MM, Nawab A, Guijarro MV, Zhang M, et al. Reprints and permission information is available at http://www.nature.com/ Oncolytic virotherapy for small-cell lung cancer induces immune infiltration and reprints prolongs survival. J Clin Invest. 2019;129:2279–92. 35. Ullman-Culleré MH, Foltz CJ. Body condition scoring: a rapid and accurate Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims method for assessing health status in mice. Lab Anim Sci. 1999;49:319–23. in published maps and institutional affiliations. 36. Kolb R, De U, Khan S, Luo Y, Kim M-C, Yu H, et al. Proteolysis-targeting chimera against BCL-XL destroys tumor-infiltrating regulatory T cells. Nat Commun. 2021;12:1281. 37. Anderson GR, Wardell SE, Cakir M, Crawford L, Leeds JC, Nussbaum DP, et al. PIK3CA mutations enable targeting of a breast tumor dependency through mTOR-mediated MCL-1 translation. Sci Transl Med. 2016;8:369ra175. Open Access This article is licensed under a Creative Commons 38. Gardner EE, Connis N, Poirier JT, Cope L, Dobromilskaya I, Gallia GL, et al. Attribution 4.0 International License, which permits use, sharing, Rapamycin rescues ABT-737 efficacy in small cell lung cancer. Cancer Res. adaptation, distribution and reproduction in any medium or format, as long as you give 2014;74:2846–56. appropriate credit to the original author(s) and the source, provide a link to the Creative 39. Thummuri D, Khan S, Underwood PW, Zhang P, Wiegand J, Zhang X, et al. Commons license, and indicate if changes were made. The images or other third party Overcoming gemcitabine resistance in pancreatic cancer using the BCL-XL- material in this article are included in the article’s Creative Commons license, unless specific degrader DT2216. Mol Cancer Ther. 2022;21:184–92. indicated otherwise in a credit line to the material. If material is not included in the 40. Lee EF, Harris TJ, Tran S, Evangelista M, Arulananda S, John T, et al. BCL-XL and article’s Creative Commons license and your intended use is not permitted by statutory MCL-1 are the key BCL-2 family proteins in melanoma cell survival. Cell Death Dis. regulation or exceeds the permitted use, you will need to obtain permission directly 2019;10:342. from the copyright holder. To view a copy of this license, visit http:// 41. Del Gaizo Moore V, Letai A. BH3 profiling-measuring integrated function of the creativecommons.org/licenses/by/4.0/. mitochondrial apoptotic pathway to predict cell fate decisions. Cancer Lett. 2013;332:202–5. 42. Fraser C, Ryan J, Sarosiek K. BH3 profiling: a functional assay to measure apop- © The Author(s) 2023 totic priming and dependencies. Methods Mol Biol. 2019;1877:61–76. Cell Death Discovery (2023) 9:1

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